Best results are observed when cells are split twice weekly. To enable a weekend-free culture schedule, cells would be split on Monday and Thursday with full media exchanges on Tuesday and Friday, approximately 24 hours after plating. In this example, the Friday feed should use twice the standard volume.

Best results are observed when cultures are switched into Gibco™ Essential 8™ Flex Medium at the start of the week, and Gibco™ Essential 8™ Flex Medium is routinely used in place of existing feeder-free culture systems.

As with many other PSC media, ROCK inhibitor is not required with Gibco™ Essential 8™ Flex Medium when Versene Solution is used to passage PSCs as clusters. If cells are plated as singlets, we suggest that Gibco™ RevitaCell™ Supplement (Cat. No A2644501) or another ROCK inhibitor be used.

Optimal results are observed when the cells are split twice weekly with a medium exchange approximately 24 hours after plating. Splitting Monday and Thursday with Tuesday and Friday (double volume) feeds have shown the best results.

The preparation of Gibco™ Essential 8™ Flex Medium is equivalent to that of the original Gibco™ Essential 8™ Medium. The only difference is the feeding schedule, with Gibco™ Essential 8™ Flex Medium enabling a more flexible, 2-day break between required culture feeds.

No, Gibco™ Essential 8™ Flex Medium is a complete kit, with a base medium and frozen supplement designed to be used together. Gibco™ Essential 8™ Flex Medium components should not be mixed with Gibco™ Essential 8™ Medium components.

No, do not thaw the Gibco™ Essential 8™ Flex Supplement in a 37°C water bath. Best results are observed thawing at room temperature for approximately 1 hour. You may also thaw the Gibco™ Essential 8™ Flex Supplement overnight at 2–8°C, a process that could result in small amounts of precipitation. The presence of precipitation should not adversely affect the performance of the medium.

The appearance of precipitate in the Gibco™ Essential 8™ Flex Supplement is rare; however, if it is seen, it should not affect the performance of the medium. If you see precipitation in the supplement, keep it well mixed and add it to the Gibco™ Essential 8™ Flex Basal Medium as you normally would.

Yes, Gibco™ Essential 8™ Flex Medium has been used with ROCK inhibitors. We suggest using Gibco™ RevitaCell™ Supplement (Cat. No. A2644501), which has been specifically designed to minimize the impact of stress on PSCs.

Best results are observed if you choose and maintain a regular split and feed schedule. For example, if you desire a weekend-free feeding schedule, you should split Monday/Thursday and feed on Tuesday/Friday.

Best results are observed when you maintain your PSCs consistently in one medium. Transitioning from Gibco™ Essential 8™ Medium to Gibco™ Essential 8™ Flex Medium is as simple as seeding your cells into Gibco™ Essential 8™ Flex Medium at the beginning of the week. Best results are achieved if the cells are split one time prior to the 2-day feed-free period.

Because optimal PSC culture results are achieved using a single PSC culture medium, we do not recommend routinely switching cells from one medium to another. Optimal results are achieved when Gibco™ Essential 8™ Flex Medium is used and maintained as the primary culture system.

Time-release products such as StemBeads™ FGF2 supplement release FGF2 into the growth medium to counteract the loss of FGF2 activity over time, while Gibco™ Essential 8™ Flex Medium prevents the loss of FGF2 activity from even happening.

As with any PSC culture, some cell debris and pH drift will occur with Gibco™ Essential 8™ Flex Medium. However, we have observed no adverse long-term effects from waste product buildup on either cell growth or pluripotency.

The potential for cells to differentiate into cells of ectoderm, endoderm, and mesoderm lineages is unaffected by long-term culture in Gibco™ Essential 8™ Flex Medium. This has been confirmed using both spontaneous differentiation from embryoid bodies and directed differentiation to neural stem cells, cardiomyocytes, and definitive endoderm.

We have seen no effect on reprogramming efficiency as compared to Gibco™ Essential 8™ Medium with Invitrogen™ CytoTune™-iPS 2.0 Sendai Reprogramming Kit. We do recommend daily feeding during colony formation (days 7–28).

Cells cultured in other feeder-free media systems, such as mTeSR™ Medium with Matrigel™ Basement Membrane Matrix, or StemPro™ hESC SFM with Geltrex™ Matrix, can be successfully cultured in Gibco™ Essential 8™ Medium and VTN-N. In addition, PSCs grown on feeders with KnockOut™ SR have also been shown to be successfully cultured in Gibco™ Essential 8™ Medium on VTN-N. However, when changing media systems, cells must be passaged either manually, or with EDTA prior to culturing on Gibco™ Essential 8™ Medium and VTN-N.

Yes. PSCs cryopreserved from cultures of mTeSR™ Medium and BD Matrigel™ Basement Membrane Matrix may be thawed into Gibco™ Essential 8™ Medium and plated on VTN-N. Certain lines may benefit from thawing into the medium and substrate they were growing in at the time of cryopreservation. Then at the next passage, use EDTA to passage the cells into Gibco™ Essential 8™ Medium and VTN-N.

You should expect to see normal pluripotent stem cell (PSC) morphology. The expected morphology of PSCs is demonstrated specifically by tightly packed colonies with defined borders and a high nucleus-to-cytoplasm ratio. See image below of PSCs at passage 6.

Other catalog versions of DMEM/F-12 cannot be used in place of the Gibco™ Essential 8™ Basal Medium in the preparation of Gibco™ Essential 8™ Medium. Gibco™ Essential 8™ Basal Medium supplied with the kit has a higher level of sodium bicarbonate.

It is very important that complete Gibco™ Essential 8™ Medium is prewarmed at room temperature and not in a 37°C water bath. bFGF activity can decline rapidly with repeated temperature changes from 4°C to 37°C.

Yes. However, this isn’t necessary, and we do not routinely use these inhibitors in house. If the use of a Rho-associated protein kinase (ROCK) inhibitor is desired, you should add the inhibitor only to the medium at passage. Inhibitors should not be present for routine feeding. Use of inhibitors is assay dependent and not required for routine cell culture.

The inclusion of either a ROCK inhibitor (HA100 or Y27632) or blebbistatin improves initial survival and supports a high cloning efficiency, which is increased by the addition of transferrin and selenium. If cells are cultured routinely in medium containing a ROCK inhibitor, it may become necessary to include it for routine culture.

Enzymes such as dispase and collagenase do not work well with cells cultured in Gibco™ Essential 8™ Medium on VTN-N. Use of these enzymes for passaging cells results in compromised viability and attachment.

The ideal time for incubation with EDTA is 4–5 minutes at 37°C. When the cells start to separate and round up, and the colonies appear to have holes in them when viewed under a microscope, they are ready to be removed from the vessel (Figure A, below). We do not recommend that you allow the colonies to break up too much, as pictured in Figure B, below.

Since EDTA has different dissociation properties than dispase and collagenase and the size of the colonies (with EDTA) is significantly smaller, the passaging ratios need to be adjusted to facilitate optimal culture conditions. Cells should be passaged when they reach ~85% confluency, which is typically at day 4. Sometimes cells will be ready for passage at day 3. Typical ratios for passaging with EDTA are 1:6, 1:8, or 1:10. Passaging ratios need to be adjusted so that cells are not ready for passaging too early or too late.

Actually, in a feeder-based culture, dispase (2 mg/mL) should take about 15–25 min to work at 37°C. Two to three minutes’ dissociation time would apply to feeder-free cultures. Dispase is a more aggressive enzyme, so it works faster, but that also means that when the PSC clumps are harvested, they are more sensitive to being broken apart by trituration. Once the clumps are harvested, they should be pipetted up and down a few times to break up the clumps to the appropriate size. If the cells are harvested with collagenase type IV, they have to be pipetted more times because the clumps are harder to break up, but this means that there is less likelihood to break up the clumps into pieces that are too small. If the cells are harvested with dispase, they have to be pipetted fewer times, and care has to be taken to ensure that the clumps are not broken too much. Either enzyme is fine to use, and if you have enough experience, you may prefer to use dispase to save time. But for a less experienced user, we recommend using collagenase type IV as it is safer and you are less likely to ruin your culture by over-triturating.

The PSC cryopreservation kit contains xeno-free PSC Cryopreservation Medium, which is a ready-to-use solution for the cryopreservation of early passage pluripotent stem cells (PSCs), and Gibco™ Revitacell™ Supplement (100X), a chemically defined recovery supplement for use in the post-thaw culture medium. When used in combination, these reagents help minimize loss of cell viability, maximize post-thaw recovery, and minimize unwanted differentiation of PSCs. This kit can also be used to cryopreserve and recover peripheral blood mononuclear cells (PBMCs) to improve post-thaw cell viability and recovery.

We have not tested this. However, the medium is stable when stored at 4 degrees C for up to 6 months. There are no components that R&D would be concerned about during a freeze thaw; however, this was not formally tested.

The optimal working concentration of rhLaminin-521 is cell line dependent and must be determined empirically. However, for some cell lines, coating concentrations as low as 0.3 μg/cm2 can be used with no decrease in performance. Additionally, coating plates overnight at 4 degrees C can support coating concentrations as low as 0.1 μg/cm2.

The formulation is the same for both products. KnockOut Serum Replacement Multi-Species is a Research Use Only product designed for use in basic research applications whereas KnockOut Serum Replacement is recommended for pre-clinical and clinical applications.

Yes. If you switch to KnockOut Serum Replacement Multi-Species for basic research applications, there will be little change to your experience with the product and it does not require regulatory documentation (or medical device clearance).

Yes, the culture vessel needs to be coated with Attachment Factor Protein (Cat. No. S006100) at 37 degrees C for 30 min or at room temperature for 2 hours. The coated vessels can be used immediately or stored at room temperature for up to 24 hours.

We recommend seeding these cells at densities ranging from 2 x 10E4 to 5.5 x 10E4 cells/cm2. A good starting point is 3 x 10E4 cells/cm2. If the feeder cells are too sparse, they may not maintain the pluripotent cells without differentiation, and the pluripotent cells may not attach well. If the feeder cells are too dense, the feeder layer may detach from the plate, and the culture will be lost.

Either method will work in arresting cell division. However, the irradiation process will ensure that cell division will cease regardless of cell aggregation. Cell clumping can potentially not inactivate all cells when using mitomycin C, as cells within clumps may not be exposed to the mitomycin C. Irradiated cells are preferred by those who have concerns about chemical treatment. Mitomycin C-treated cells are preferred by those who have concerns about DNA damage from irradiation.

Yes. Following reconstitution, complete StemFlex Medium can be aliquoted and stored at -5 to -20 degrees C for up to 6 months. Alternatively, aliquots of the supplement can be made and frozen at -5 to -20 degrees C for up to 6 months. Avoid multiple freeze-thaw cycles.

The use of ROCK inhibitor is not required when culturing in StemFlex Medium on rhLaminin-521. However, supplementation with RevitaCell Supplement can provide additional support to PSCs during stressful transitions such as single-cell passaging.

The formulation of StemFlex medium does include BSA and thus is not considered xeno-free. It is more defined than mTeSR1 as it contains less material in the formulation. Essential 8 and Essential 8 Flex are recommended for the most defined and xeno-free PSC culture media.

StemFlex Medium is a more robust medium than TeSR-E8 but has fewer components than mTeSR. Performance of StemFlex Medium was shown to be superior to mTeSR1 in a number of applications including single-cell passaging, gene editing and reprogramming.

This transition is very straightforward. We recommend at least a 2-passage transition into the StemFlex Medium system. Briefly, if you have cryopreserved cells previously cultured in the mTeSR1/Matrigel system, we recommend thawing the cells back into mTeSR1/Matrigel until fully recovered. Upon reaching ~70-85% confluency, passage using Versene solution and seed directly into the StemFlex Medium system.

Yes. PSCs cryopreserved from cultures of other media systems may be thawed directly into the StemFlex Medium system. However, certain lines may benefit from thawing into the medium and substrate they were growing in at the time of cryopreservation. Then at the next passage, use Versene solution to passage the cells into the StemFlex Medium system.

We recommend use of the Neon Electroporation device for electroporation of PSCs with Cas9 protein:guide RNA complex following the protocol guidance in the following demonstrated protocol; see the section entitled “Knockout by electroporation of RNP using the Neon Transfection System.” We have seen that the Neon electroporation protocols 7 and 14 provide optimal indel formation while maintaining cell survival. However, the electroporation conditions may need to be optimized for your pluripotent cell line.

We have not yet evaluated the Geltrex matrix system for clonal expansion in the presence of StemFlex Medium. However, rhLaminin-521 does provide optimal survival of cells following single-cell passaging and thus this matrix is recommended for such critical applications.

Yes. If improved reprogramming efficiency is required, then you may utilize Essential 7 (Essential 6 + bFGF) or Essential 8 Medium for reprogramming somatic cells and directly transition them into StemFlex Medium system upon colony selection.

We have evaluated the use of Cytotune-iPS 2.0 Sendai Reprogramming Kit (Cat. No. A16517) for somatic cell reprogramming of both neonatal and adult human dermal fibroblasts. For fibroblasts, follow the instructions provided in the Cytotune 2.0 reprogramming manual for feeder-free reprogramming (pgs. 16-20). On Day 7, you may use rhVTN-N (Cat. No. A14700), Geltrex matrix (Cat. No. A1413302), or rhLaminin-521 (Cat. No. A29248 or A29249). From Day 8 onward, rather than feeding daily with Essential 8 Medium, we recommend that you feed reprogrammed fibroblasts every-other-day with StemFlex Medium.

We have tested neural stem cells (NSCs) isolated from fetal tissue or derived from pluripotent stem cells (PSCs), and have seen that both populations can benefit from maturation medium (DMEM/F12 + Dopaminergic Neuron Maturation Supplement) to have nicely spread homogenous neurons with reduced progenitor population. Matured neurons can be further maintained in neurobasal medium supplemented with Dopaminergic Neuron Maturation Supplement.

Our studies have shown that the confluency of cells when starting differentiation can have a dramatic impact on efficiency of differentiation. Therefore, we recommend a range finding study to determine optimal confluency of each PSC line when starting differentiation. Guidance for this can be found in the product insert. Additionally, we recommend singularizing cells prior to differentiation rather than passaging in small clumps. Singularizing PSCs for differentiation to cardiomyocytes allows better seeding and confluence estimates, resulting in more consistent results well-to-well and overall better differentiation of difficult-to-differentiate lines.

It is critical to use high-quality human PSCs (with minimal or no differentiated colonies) that are karyotypically normal, confirmed to exhibit pluripotency markers, and are undergoing routine culture with regular subculture intervals and maintaining healthy morphology before starting cardiomyocyte differentiation. Additionally, we recommend that a PSC line not be used past 100 passages.

Yes. Variability is normal and it is not uncommon to find certain lines that will not differentiate as efficiently. Including a control line, such as the human ESC H1 or H9 cells, which have been shown to differentiate consistently well, may be helpful.

We recommend using EDTA for passaging PSCs. For seeding cells for definitive endoderm induction, we recommend using StemPro™ Accutase™ Cell Dissociation Reagent for small clumps and Gibco™ TrypLE™ Enzyme for singularized cells.

iPSCs are genetically reprogrammed somatic cells that exhibit a pluripotent stem cell–like state similar to embryonic stem cells. iPSCs can be derived by inducing selected gene expression via various methods including virus-mediated gene transduction and chemical induction.

We offer the Invitrogen™ CytoTune™™-iPS 2.0 Sendai Reprogramming Kit (Cat. Nos. A16517, A16518) which is a non-integrating systems that uses Sendai virus vectors to reprogram somatic cells into induced pluripotent stem cells (iPSCs). The Invitrogen™ CytoTune™-iPS 2.0 Sendai Reprogramming Kit contains three Invitrogen™ CytoTune™ 2.0 reprogramming vectors that are used for delivering and expressing key genetic factors necessary for reprogramming somatic cells into iPSCs. Only one application of the vectors is required for successful reprogramming.

Additionally, we offer the Episomal iPSC Reprogramming Vectors, also a non-integrating system that reprograms somatic cells into induced pluripotent stem cells (iPSCs). This product is a mixture of three vectors designed to provide the optimal system for generating transgene-free and virus-free iPSCs in a feeder-free environment. Originally developed by Junying Yu and James Thomson and further optimized by Cellular Dynamics International, these Episomal iPSC Reprogramming Vectors have proven successful in reprogramming a number of different somatic cell types.

The Invitrogen™ CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Cat. Nos. A16517, A16518) is a non-integrating system that uses Sendai virus vectors to reprogram somatic cells into induced pluripotent stem cells (iPSCs). The Invitrogen™ CytoTune™-iPS 2.0 Sendai Reprogramming Kit contains three Invitrogen™ CytoTune™ 2.0 reprogramming vectors, including the four Yamanaka factors, Oct3/4, Sox2, Klf4, and c-Myc. The expression of these transcription factors in somatic cells has been shown to be a critical factor in the successful generation of iPSCs. Only one application of the vectors is required for successful reprogramming.

Yes we did, and found that only the combination of KOS (3 in 1), cMyc, and Klf4-vector yields highest reprogramming efficiency. For instance, KOS and cMyc alone are not sufficient for reprograming. Addition of Oct4 or Sox2 results only in a very few reprogrammed colonies. This is mostly due to an imbalance in the stoichiometry of the reprogramming factors, which may impair the reprogramming efficiency significantly.

The Invitrogen™ CytoTune™ -iPS Sendai Reprogramming Kit (Cat. Nos. A13780-01, A13780-02) is a non-integrating system that reprograms somatic cells into induced pluripotent stem cells (iPSCs). This kit utilizes four Sendai virus-based vectors, each capable of expressing one of the four Yamanaka factors: Oct3/4, Sox2, Klf4, and c-Myc. The expression of these transcription factors in somatic cells has been shown to be a critical factor in the successful generation of iPSCs.

The 2.0 kit contains three vectors, one of which is a polycistronic vector (Invitrogen™ CytoTune™ 2.0 KOS), designed to deliver increased reprogramming efficiency. This polycistronic vector has a different backbone containing temperature-sensitive mutations in the polymerase-related genes, and this helps to clear the virus faster after reprogramming and causes less cytotoxicity to the cells.

The polycistronic configuration of KOS requires that additional polymerase activity be available to compensate for the combination of three genes on one vector. The Invitrogen™ CytoTune™-iPS 2.0 system uses the extra polymerase from the hKlf4 vector to drive reprogramming in all vectors and enhance reprogramming efficiency. In addition, increased expression of hKlf4 also enhances reprogramming efficiency.

Yes, the additional Klf4 vector allows the system to be fine-tuned by the user. The amount of Klf4 can be increased to enhance reprogramming efficiency, or decreased to minimize the total amount of virus.

The reprogramming vectors from the Invitrogen™ CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Cat. Nos. A16517, A16518) are not compatible with the reprogramming vectors from the original Invitrogen™ CytoTune™-iPS Sendai Reprogramming Kit (Cat. Nos. A13780-01, A13780-02). Do not mix or substitute Invitrogen™ CytoTune™ 2.0 reprogramming vectors with the reprogramming vectors from the original kits.

Sendai virus, also known as Hemagglutinating Virus of Japan (HVJ), is a respiratory virus of mouse and rat first isolated in Sendai, Japan in the early 1950s. The virus is classified as mouse parainfluenza virus type I, belonging to the Paramyxoviridae family. SeV is an enveloped virus, 150–250 nm in diameter, whose genome is a single chain of (–) sense RNA (15,384 bases). The virus infects cells by attaching to the sialic acid receptor present on the surface of many different cells and is thus able to infect a wide range of cell types of various animal species.

The Sendai virus vectors in the Invitrogen™ CytoTune™-iPS 2.0 Sendai Reprogramming Kit are based on a modified, non-transmissible form of SeV, which has the fusion protein gene (F) deleted. The viral vectors maintain full infectivity to a wide range of cells; however they are no longer capable of producing infectious particles from infected cells because the viral genome lacks the F gene. The Sendai virus vectors contain transgenes that will express factors hOct3/4, hSox2, hKlf4, and hc-Myc. After transduction, the viral vectors will cause the cells to express these four genes, resulting in reprogramming.

The main difference between Sendai virus and lentivirus is that, when using SeV reprogramming methods, the vectors and transgenes can be eliminated from the cells. Some viruses, such as lentiviruses, require integration of viral DNA into the host genome. This can be problematic because this integration is random and can potentially disrupt the function of important genes. Sendai virus requires no integration for viral proteins to be made in the host cell. Other DNA-based viruses, like adenovirus, are non-integrating but must localize inside the nucleus for the viral proteins to be made. This means that there can still be random integration events, where the viral DNA integrates into the host genome. Since Sendai virus is an RNA virus, it does not need to enter the nucleus for transcription. This eliminates the possibility of integration of the transgenes into the host genome.

Integration-free reprogramming methods generate iPSCs that do not contain detectable vectors or transgenes. Traditional technologies used for reprogramming (e.g., lentivirus, retrovirus) integrate into the genome of the target cells. The resulting iPSCs and cells differentiated from those iPSCs will contain foreign DNA and could be unsafe and problematic for use in cell therapy and drug discovery applications. Furthermore, the integration could occur in a critical region of the genome, causing problems with unrelated developmental processes.

Our reprogramming kits have been validated for a wide variety of cell types, including human fibroblasts, CD34+ cord blood cells, and peripheral blood mononuclear cells (PBMCs). For a current list of publications citing the cell types validated using this method, go here.

Although humans are not a natural host for SeV, and the virus is non-pathogenic to humans, appropriate care must be taken to prevent the potential mucosal exposure to the virus. The Invitrogen™ CytoTune™-iPS 2.0 Sendai Reprogramming Kit must be used under Biosafety Level 2 (BL-2) containment with biological safety cabinet and laminar flow hood, and with appropriate personal safety equipment to prevent mucosal exposure/splash.

The virus does get passed on to daughter cells, and gradually over time its concentration diminishes. The virus can’t leave the cells and infect new cells though, because the fusion gene has been deleted from the viral genome.

One kit (one tube of each vector, i.e., 3 tubes) is sufficient for a minimum of 5 wells of a 6-well dish at MOI of 5:5:3 (KOS, hc-Myc, hKlf4) with a recommended plating density of 2 x 10E5 to 3 x 10E5 cells/well for human dermal fibroblasts. The virus can only be used once, as viral titers decrease significantly with each freeze-thaw cycle.

One kit (one tube of each vector, i.e., 4 tubes) is sufficient for 2 wells of a 6-well plate (5 x 10E5 cells/well) at MOI = 3. The kit is available in two sizes: Cat. No. A13780-01 contains 1 tube of each vector (total 4 tubes), while Cat. No. A13780-02 includes 3 tubes of each vector (total 12 tubes). The virus can only be used once, as viral titers decrease significantly with each freeze-thaw cycle.

This can vary depending on the cell type. We recommend the following for initial experiments: one to two days before transduction, plate your cells onto two wells of a 6-well plate at the appropriate density to ensure that the cells are 80–90% confluent the day of transduction. Since overconfluency results in decreased transduction efficiency, we recommend replating your cells to achieve 80–90% confluency if your cells have become overconfluent during culturing.

MOI (multiplicity of infection) describes the ratio of viral particles to cells. The three vectors in the Invitrogen™ CytoTune™ 2.0 Kit should each be added to the cells based on an MOI recommendation. We recommend the following MOIs as a starting point, and adjustments can be performed if reprogramming efficiency is not optimal.

An MOI of 5:5:3 (KOS, hc-Myc, hKlf4) is recommended for most cell types. The virus titer varies from lot to lot; the required volume for each MOI is listed on the Certificate of Analysis (CoA) for each lot of product. You may also choose to optimize your MOI as this may vary depending on the cell type. The ratio of KOS and hc-Myc must be 1 to 1, and the MOI of hKlf4 can be varied independently. For example: if KOS is 4, then hc-Myc must also be 4.

We recommend first to try increasing the MOI of hKlf4 only. For example: go from 5:5:3 to 5:5:6. If optimization is still required, then increase the MOI of KOS and hc-Myc. The ratio of KOS and hc-Myc must be 1 to 1, and the MOI of hKlf4 can be varied independently. For example: go from 5:5:3 to 10:10:3 or 10:10:6.

An MOI of 3 is recommended for most cell types. The virus titer varies from lot to lot; the required volume for an MOI of 3 is listed on the Certificate of Analysis (COA) for each lot of product. You may also choose to optimize your MOI as this may vary depending on the cell type.

Yes. Initial experiments with fibroblasts have shown that scaling down to a 12-well or 24-well culture dish works, but at a potentially reduced efficiency. Cell seeding densities may need to be optimized.

If you want to reprogram PBMCs with Invitrogen™ CytoTune™ 2.0 under feeder-free conditions, you should follow the existing Invitrogen™ CytoTune™ 2.0 PBMC protocol, but plate onto Gibco™ Vitronectin or Geltrex™ matrix on day 3 instead of MEF, and then transition over to Gibco™ Essential 8™ on Days 7–8 instead of Gibco™ KnockOut™ Serum Replacement–based PSC medium.

Efficiencies are typically lower than with feeder-dependent conditions, but you should still get some colonies.

The reprogrammed cells can be grown on feeders in a Gibco™ KnockOut™ Serum Replacement (KSR) complete medium in a feeder-dependent culture, or feeder-free in Gibco™ StemPro™ hESC SFM. For KSR medium, we recommend the following. To prepare 100 mL of Human iPSC Medium, aseptically combine the components listed below:

Component

Stock concentration

Final concentration

Volume

KnockOut™ DMEM/F-12 (Cat. No. 12660-012)

—

1X

78 mL

KSR (Cat. No. 10828-028)

—

20%

20 mL

MEM Non-Essential Amino Acids Solution (Cat. No. 11140-050)

10 mM

0.1 mM

1 mL

GlutaMAX™-I (Cat. No. 35050-061)

100X

1X

1 mL

β-mercaptoethanol, 1000X (Cat. No. 21985-023)

1000X

1X

100 µL

Penicillin-Streptomycin (optional) (Cat. No. 15140-122)

100X

1X

1 mL

bFGF* (Cat. No. PHG0264)

10 µg/mL

4 ng/mL

100 µL

*The medium can be stored at 2–8°C for up to one week. Add bFGF when the medium is used.

The reprogrammed cells can be grown in standard iPSC culture medium. We recommend either Gibco™ KnockOut™ Serum Replacement (KSR)–supplemented medium in a feeder-dependent culture, or feeder-free in Gibco™ Essential 8™ Medium. Refer to the user manual for the full protocol.

iPSC colonies will begin to form roughly 3 weeks post-transduction. Only one application of the vectors is required for successful reprogramming, enabling selection of iPSC colonies 21–28 days after transduction.

The Invitrogen™ CytoTune™-iPS 2.0 Sendai Reprogramming Kit offers reprogramming efficiencies in the range of 0.02– 1.2% with BJ fibroblasts. This may vary for other cell types. Please go here for more information.

The Invitrogen™ CytoTune™-iPS Sendai Reprogramming Kit (discontinued) provides a 100–fold increase in efficiency over lentiviral methods to generate iPSCs, allowing for reprogramming efficiencies between 0.01% and 1%. Please see the table below for comparison of efficiencies with various reprogramming methods.

The expected morphology of iPSCs is demonstrated specifically by tightly packed colonies with defined borders and a high nucleus-to-cytoplasm ratio, as seen in the figure below. If you do not observe this morphology or the number of colonies observed is low, then the MOI used for transduction may need to be increased.

The iPSC colonies can be easily visualized using alkaline phosphatase stain, such as the Alkaline Phosphatase Live Stain (Cat. No. A14353). See image below. In addition, reprogrammed colonies can be selected utilizing live staining with Tra1-60 or Tra1-81 antibodies that recognize undifferentiated iPSCs and enable the identification of reprogrammed cells from a variety of human cell types. Refer to the user manual for the full protocol.

We have used this combination of primary and secondary with no problems. IgG secondary antibodies will cross-react with IgM primary antibodies, since IgM share the same kappa light chains as IgG.

The IgG secondary antibodies that are listed in manuals are available, and they will work against IgG primary antibodies, as well as IgM primary antibodies, and others. For that reason, we typically have those IgG secondary antibodies on hand, and use them in most applications. That is the main reason it was recommended.

iPSCs must be monitored and growth medium must be replaced daily in order to maintain a healthy culture. In general, iPSC colonies should be passaged when the cells reach 70–80% confluence or when most of the colonies are larger than 700 μm. Refer to the user manual for the full protocol.

It can take as few as five or as many as fifteen passages for the virus to clear from the cell. In rare cases Sendai sequences can persist indefinitely. Clearance rate is clone-dependent and can be confirmed by PCR or by anti-Sendai antibody. For more information about generating vector-free iPSCs, please refer to the user manual.

It typically takes 5–10 passages for the virus to clear from the cell. This can be confirmed using several methods including staining with anti-Sendai virus antibody or performing RT-PCR using the TaqMan™ -iPSC Sendai Detection Kit (Cat. No. A13640), see page 17 of the Invitrogen™ CytoTune™ -iPS Sendai Reprogramming Kit manual. The data below demonstrate the absence of the virus in iPSCs generated using the Invitrogen™ CytoTune™-iPS Sendai Reprogramming Kit.

We typically make working solutions of 2 mg/mL, and we do not take the units per mg into account that differ from lot-to-lot. We are aware that different dispase batches may have different enzymatic activity but so far we have not encountered any problems using this method. It is important to not incubate the cells too long in dispase, this enzyme works quickly, and incubating too long can lead to clump sizes that are too small. So as long as you keep incubation to around 2–3 minutes, there should be no problem, even if the enzymatic activity may vary a bit from batch to batch.

The dispase manual does not specifically include information about its use with PSC (pluripotent stem cell) culture, so that is why there is a difference between the manual and the PSC protocol.

The Invitrogen™ CytoTune™-EmGFP Sendai Fluorescence Reporter is a fluorescent control vector carrying the EmGFP gene. The fluorescent control vector allows the determination of whether a cell line of interest is amenable or refractive to infection by Sendai reprogramming vectors.

Add the reporter one time to your cells and monitor for expression. Different cell types may vary in their ability to take up Sendai virus; therefore, we suggest initially transducing your cells with at least 2–3 different MOIs (e.g., 1, 3, and 9). Please refer to the user manual for the full protocol.

If you want to use the EmGFP Reporter with reprogramming, it must be added at the time of reprogramming. Cells infected with Sendai virus will most likely be refractive to further infection. Therefore, do not try to add Invitrogen™ CytoTune™-iPS 2.0 Sendai Reprogramming Kit to cells already transduced with Invitrogen™ CytoTune™-EmGFP Sendai Fluorescence Reporter or vice versa.

You can confirm the presence of the Sendai virus in your cells by several methods, including staining with anti-Sendai virus antibody or performing RT-PCR using TaqMan™-iPSC Sendai Detection Kit (Cat. No. A13640), see page 17 of the Invitrogen™ CytoTune™-iPS Sendai Reprogramming Kit manual.

For the Invitrogen™ CytoTune™ 2.0 kit, which contains 3 vectors (KOS, cMyc, Klf4), both the cMyc and the KOS vector can be cleared by a temperature shift to 39°C. The temperature shift will be most effective for the cMyc vector, but should still work for the KOS vector.

Note: Before performing a temperature shift, first verify that the individual Klf4 vector has cleared. The Klf4 vector will not be cleared by a temperature shift, and performing the shift while the Klf4 is still present could cause problems.

For the original Invitrogen™ CytoTune™ kit, which contains 4 vectors (Oct4, Sox, Klf4, cMyc), the cMyc vector is the only one that can be cleared by the temperature shift to 39°C. The other three vectors remain active at 39°C.

No, we do not have such primers. However, an alternative method could be to use the TaqMan™ iPSC Sendai Detection Kit (Cat. No. A13640), which will indicate the presence of the Invitrogen™ CytoTune™ vectors in the iPS cells. If Invitrogen™ CytoTune™ vectors are detected, then you will need to keep passaging the cells. If there is no detection of Invitrogen™ CytoTune™ vectors, then you can look at expression of the reprogramming genes, and be sure that any expression that is seen is solely due to endogenous expression, and not from the vectors. Of course, this only applies to established iPSC. To study activation of the endogenous genes during reprogramming, the full sequence info of the vectors would be required (but that information is proprietary).

The TaqMan™ iPSC Sendai Detection Kit was designed for Version 1 of the Invitrogen™ CytoTune™ kit, but there is some overlap between the Version 1 and Version 2 vectors. We recommend that customers use qPCR and the SeV backbone primer set to look for overall viral clearance in the Invitrogen™ CytoTune™ 2.0 kit, and if they need to look at the presence of specific vectors, they should perform endpoint PCR and use the primers listed in the manual. Those primers are not suitable for qPCR. The SeV backbone is available as an individual TaqMan primer set.

iPSCs cultured on MEF feeder layers can be adapted to feeder-free conditions in Gibco™ StemPro™ hESC SFM. This can be done by directly thawing or splitting the iPSCs in MEF-conditioned medium (MEF-CM) and then replacing the culture medium daily with medium that contains increasing amounts of StemPro™ hESC SFM. For the complete protocol, visit our web protocols.

The L-Myc virus was chosen over the c-Myc virus because it is considered superior from a safety perspective, for clinical applications (L-myc is reported to have lower transformation/oncogenic potential than c-myc).

It is more expensive because of its label use statement: For research use and non-commercial manufacturing of cell based products for clinical research. CAUTION: Not intended for direct administration into animals or humans. The kit is manufactured according to GMP requirements and the drug master file is available for submission to FDA upon customer request, for use in clinical research.

Gibco™ StemPro™ Neural Stem Cells (Cat. No. A15654 or A15655): Isolated from human fetal cortex brain and manufactured under good manufacturing practice (GMP). Each lot is generated from the same master bank (same donor) so that the lot-to-lot variability is low. Cell doubling time is ~100 hours.

Both cells are tested for their ability to retain their proliferation and differentiation potential for at least 3 passsages after thawing. Both are able to differentiate into neurons, astrocytes, and oligodendrocytes. However, StemPro™ Neural Stem Cells are recommended to grow in suspension culture as adherent culture would trigger differentiation, whereas Human Neural Stem Cells (H9-derived) can grow under both suspension and adherent conditions.

We do not recommend using Gibco™ StemPro™ NSC SFM to expand NSCc which are induced using Gibco™ PSC Neural Induction Medium (Cat. No. A1647801), because the morphology of NSCs induced from some lines of hPSCs will change if this medium is used to expand NSCs. We recommend using the Neural Expansion Medium to expand such NSCs, as stated here.

These cells can be thawed and passaged once before using in experiments. The single passage will yield a 2 fold increase of thawed cells. Cells do not expand significantly beyond first passage post-thaw.

We offer Gibco™ Hibernate™ Medium for this purpose. When supplemented with Gibco™ B-27™ Supplement and Gibco™ GlutaMAX™-1 Supplement, this medium can allow for the manipulation of neurons at ambient CO2 for at least 48 hours while retaining their viability, and preserve viable brain tissue for up to a month when stored at 4°C. Two Hibernate™ media are offered:

L-glutamate is excitotoxic as neuron cells mature. For primary hippocampal neurons and other embryonic neurons, we recommend that you add 25 µM L-glutamate to the initial plating medium. However, after day 4, L-glutamate should not be added as it is toxic to neuron cells beyond day 4.

For neuroblastomas, L-glutamate should be included in the medium for both plating and subsequent medium changes.

The Gibco™ B-27™ Physiology Kit is optimized for electrophysiology experiments, and developed to enhance network spike rates. It contains extra components that help to increase spike rate and enhance synaptogenesis.

It is better to treat differentiating neurons continuously with CultureOne Supplement. You can withdraw CultureOne Supplement after 2 weeks of differentiation; however, withdrawal of CultureOne Supplement may increase the chances of clumps reforming in the culture.

Stem Cell Staining

AP is a phenotypic marker of pluripotent stem cells (PSCs), including undifferentiated embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), embryonic germ cells (EGCs) and Embryonic Carcinoma Cells (ECCs). While AP is expressed in most cell types, its expression is highly elevated in PSCs. Therefore, AP staining has been used to differentially stain PSCs to easily distinguish them from mouse embryonic fibroblasts (MEFs) used as feeders and parental fibroblasts commonly used in reprogramming experiments.

The Alkaline Phosphatase Live Stain is a stem cell imaging product that allows users to differentially stain pluripotent stem cells (PSCs). The AP Live Stain utilizes an easy, non-permanent, cell viable protocol for identifying PSCs in your experiments. The stain is provided as a concentrated solution that is diluted in basal medium prior to adding to cells.

The dye is a cell-permeable fluorescent substrate for alkaline phosphatase (AP) that is non-toxic to cells, diffusing out over the course of two hours. Simply dilute the dye in basal medium, apply to cells, gently wash, and the cells are ready for fluorescent imaging.

Unlike traditional alkaline phosphatase staining assays, which are terminal, the Alkaline Phosphatase Live Stain allows you to visualize your pluripotent stem cell colonies without destroying your cells. The alkaline phosphatase substrate in the Alkaline Phosphatase Live Stain is non-toxic to cells and diffuses out in two hours.

There are a few key usage steps around removing the growth medium, diluting the stain, and washing the cells. We recommend this short video before use. Alternatively, the subsequent AP Live Stain FAQs address these key points.

Remove the AP Live Stain vial from the –20°C freezer and thaw at room temperature. Avoid repeated freeze/thaw cycles and aliquot if necessary. Maintain the stock solution and aliquots protected from light in amber tubes and minimize exposure to atmospheric conditions. To prepare a 1X AP Live Stain working solution, dilute the 500X stock solution in DMEM/F-12 (Cat. No. 10565-018). Use the diluted dye immediately.

Do not add the concentrated 500X stock solution directly to the dish. AP Live Stain must be diluted 1:500 in basal medium, such as DMEM/F-12 (Cat. No. 10565-018). Prior to adding AP Live Stain, the growth medium must be removed, and the cells must be gently washed twice with pre-warmed basal medium. The 1X AP Live Stain solution is then added directly onto the adherent cell culture.

There are two washing steps in the staining procedure. First, the cells are washed twice after the removal of the growth medium. Then, after the cells are stained and the dye is removed, the cells are washed twice to eliminate the excess AP Live Stain and reduce the background signal. Washing should be performed gently with pre-warmed basal medium, such as DMEM/F-12 (Cat. No. 10565-018). Handle the cells aseptically, and carefully add and remove the medium with minimal disruption to the adherent cells during this step to avoid damage to cells.

It is important that the washes are performed gently, but effectively, to remove excess substrate. Tipping the dish gently and adding the medium to the corner of the dish rather than directly onto the cells will help maintain viability. This same technique should be used for removal of medium and the subsequent wash steps. It is important to use medium that is sterile and has been pre-equilibrated to 37°C and is at the proper pH to ensure cell survival.

After 20–30 minutes, remove the AP Live Stain and wash the cells twice for 5 minutes each with DMEM/F-12 (Cat. No. 10565-018). Following the final wash, add fresh DMEM/F-12 and visualize the stained colonies under fluorescent microscopy using a standard FITC filter. Images should be captured within 10 to 30 minutes following the removal of the dye and the most robust fluorescent colonies should be marked for selection and expansion. Since the fluorescent signal leeches out of the cells and into the surrounding medium as the stain is turned over, we strongly encourage that you visualize and capture images immediately following the final wash for optimal signal detection.

AP Live Stain is ideal for screening colonies during early stages of reprogramming since it selectively stains PSCs while maintaining cell viability. It can be used in later stages of reprogramming to identify undifferentiated cells for the selection of iPSCs for further cultivation. AP Live Stain was developed specifically for use on live cultures for cell maintenance and is qualified to be free of mycoplasma and bioburden, and exhibits extremely low endotoxin levels.

Cells can be co-stained with AP Live Stain and any non-FITC labeled antibody. We recommend, however, that the staining occurs sequentially; stain with surface marker antibodies and appropriate non-FITC fluorophores prior to staining with AP live stain since the antibody staining is semi-permanent and will last for several hours, but AP live stain is transient. Note that although AP Live Stain is rigorously qualified for use in live staining, the surface marker antibodies may not be, and thus PSCs that are co-stained with antibodies may not maintain cell viability and sterility.

Store AP Live Stain at –20°C in the freezer and thaw at room temperature. Avoid repeated freeze/thaw cycles and aliquot the AP Live Stain into smaller volumes if necessary. Always protect from light and avoid extended exposure at room temperature and atmospheric conditions.

Since AP Live Stain depends on differential expression of alkaline phosphatase, dim staining of mouse embryonic fibroblasts (MEFs) may be observed. Observe the entire dish to distinguish PSC colonies from individual MEFs that may have high levels of autofluorescence. This will not interfere with your experiment. See image below for an example of this type of staining.

Serum or serum replacement components in the growth medium may cause background and this can result in poor or dim staining. After the removal of the growth medium, gently wash the culture with pre-warmed DMEM/F-12 (Cat. No. 10565-018) for 5 minutes. Aspirate and repeat once before adding the AP Live Stain. To further decrease background staining, perform 3 separate washes of 5 minutes each with DMEM/F-12 for a total of 15 minutes following the staining, and visualize immediately.